Prion diseases are a focal point of public interest, recently fuelled by the detection of unexpected cases of CJD in teenagers and in farmers, both in Great Britain, where transmission of prion proteins from cattle to humans via meat consumption is postulated, thus indicating the transmission of BSE to humans, thereby causing CJD.
Several factors enhance public concern:    a) the nature of the causative agent, the so called prion protein, of SEs is unknown or at least controversial,    b) whatever its nature, the agent is highly resistant to procedures that eliminate other infectious agents (e.g., heating);    c) therapeutical interventions are apparently not possible, once symptoms occur;    d) SEs have an extremely long incubation period;    e) practical, sensitive and specific diagnostic methods to be used during the preclinical phase are not available. This all adds to the general feeling of “living with a time-bomb”. Not only the possible presence of prion proteins in meat and neat products poses a health threat, also the possible presence of prion proteins in blood and blood products used in transfusion, the presence in pharmaceutical products of animal origin, in cosmetics of animal origin, in sera used for cell culture, in short, in an extensive array of products of animal origin, pose possible threats to human and animal health.
Until now, confirmatory diagnosis of scrapie and also other transmissible spongiform encephalopathies depended on histological examination of the brain, collected during post-mortem examination from animals or humans with clinical signs of the disease. Deposits of an aberrant or altered protein (PrPSc, prion protein) can be detected in the brain of diseased animals. This protein is very insensitive to methods, such as proteinase K digestion, that otherwise denature, lyse or remove normal proteins. The aberrant protein is considered central in the pathogenesis of prion disease. Albeit not infectious in a classical microbiological way due to the absence of specific nucleic acid, the aberrant protein itself is seen as the causal agent, and when a susceptible animal obtains such an aberrant protein in its body (i.e. by ingestion, inoculation or via mutation of the gene of the normal version of the PrP protein, PrPC) a chain reaction may start that ultimately will lead to a clinical manifestation of prion disease. The chain reaction entails the formation of more aberrant proteins formed out of the normal protein present in the animal's body. Normal and aberrant forms will interact in such a way that more aberrant forms are produced. Since the aberrant form is very resistant to proteolysis, deposits of the converted prion protein will be formed, especially in the brain and other parts of the central nervous system (CNS), giving rise to the spongiform encephalopathy and thus clinical manifestations of brain disease.
As SE-infected or affected animals and man lack a disease-specific immune response, identifying individuals before they develop clinical signs (which can take years) has been practically impossible so far. No biochemical, haematological, or gross pathological abnormalities are consistently associated with SEs. The diagnosis of SEs, therefore, depends on the recognition of clinical signs, electro-encephalography or magnetic resonance imaging techniques (both used only in human patients), or the more invasive method of taking brain-biopsies. The final diagnosis is made during autopsy, by histological examination of the brain. The neuro-pathological lesions, consisting of vacuolation (spongiform change) of the grey matter associated with gliosis and neuronal loss, are generally sufficiently characteristic. Further confirmation is possible by demonstrating scrapie associated fibrils (SAFS) in brain extracts, or by demonstrating the presence of its constituent protein, PrPSc. PrPSc is associated with the disease and is an aberrant form of the host encoded prion protein (PrP), the aberrant form is induced by a conformational change. PrPSc can be detected by immunological techniques such as Western blotting or immunohistochemistry. The latter technique is gradually becoming more and more accepted as a reliable diagnostic tool for clinical cases, in both the human and veterinary SE field.
The search for a practical preclinical diagnostic test has been and continues to be a main topic of research. This generally focuses on the detection of infectivity using a bio-assay, or the detection of the disease associated PrPSc. The bio-assay, in spite of being the most sensitive detection method, is far too cumbersome and time-consuming to ever become a practical diagnostic method: test results might become available long after the patient has passed away.
Most researchers have therefore focused on techniques to detect PrPSc. Although not all researchers agree with the statement that PrPSc is the causative agent, most, if not all, agree that the association of the presence of PrPSc and disease has been firmly established. Detection of PrPSc in tissues outside the CNS would allow sampling through less invasive methods than brain biopsies, thereby brightening prospects for a practical preclinical diagnostic technique substantially. Various tissues have been used in an attempt to develop an early detection technique: blood, urine, tissue fibroblasts, and, particularly in the animal field, lymphoid tissue. A short summary of the most promising and striking ones is given here (for an extensive review see Schreuder, 1994a, 1994b ).
Blood: In human SEs, there is the often disputed experimental transmission of CJD from buffy coat samples of human CJD-patients to rodents (Muaramoto et al., 1993), but there is little or no indication that blood and specifically, buffy coat contains any infectivity in animals affected naturally with scrapie, either in clinical or in preclinical stages (Fraser and Dickinson, 1978; Hadlow et al., 1982). Interesting results have recently been reported by Meiner et al. (1992) who detected PrPSc in peripheral tissues, both in cultured fibroblasts and in monocytes, in a group of eight CJD patients carrying the codon 200 mutation and suffering from clinical disease. These authors used both Western blotting and immunocytochemistry techniques. Their publication, however, appears to have had no follow-up and even if these results could be confirmed, the chances for a reliable blood test seem remote, at least in the case of animal SEs and given the number of negative reports from literature (reviewed in Brown, 1995).
Urine: Only once has a claim been made that infectivity in urine was demonstrated in a case of CJD, by transmitting it to mice. The same author was, however, unable to repeat this experiment (Brown, 1995). A totally different approach was reported recently (Brugere et al., 1991). Urine from scrapie affected and control animals was tested in a voltametric method by repeated capillary micro-electrolysis, which allowed discrimination of these two groups. This approach appeared promising, but, its value in detecting preclinical stages of in particular BSE could not be confirmed.
Lymphoid tissue: Lymphoid tissue has apparently not been used in the field of diagnosing human SEs, it has, however, in the veterinary field. The already classical work by Hadlow has shown that in the lymphoid tissue of naturally infected scrapie sheep, infectivity was detectable by bio-assay as early as 10–14 months of age. This was before any infectivity in the CNS was found (Hadlow et al., 1980). Western blotting has revealed the presence of PrPSc in the spleen of scrapie-infected mice (Diringer et al., 1983; Doi et al., 1988), in some cases PrPSc was detected as early as 4 weeks after experimental infection. Pooled lymph nodes from these mice also contained PrPSc. Similarly, also using Western blotting, PrPSc was detected fairly consistently in a group of naturally injected sheep showing clinical signs of scrapie, in samples from the CNS, spleen, and lymph nodes (Ikegami et al., 1991). The value of this Western blotting technique was, at least for clinical cases, confirmed by other groups. The results, however, from a group of experimentally infected sheep that were killed at 16, 18 and 21 months after inoculation but before clinical signs developed, were inconsistent and difficult to evaluate: PrPSc was detected in spleen samples of only 3 out of 12 supposedly positive animals, with lymph node samples only weak or doubtful results, but no positive results were found, illustrating the insensitivity of this technique. Therefore, using Western blotting techniques in pre-clinical diagnoses of TSE give erratic and not reliable results.
The reason for these erratic results can be found in the method to prepare the PrPSc protein (present in the affected tissues) and dissociate or separate it from the normal cellular isoform PrP protein that is also immunoreactive with the same antisera used for the Western blotting.
Ikegami et al. (1991) and Muramatsu et al. (1993) need to prepare the samples for Western blot analysis by various steps. They first enrich the samples by preparing tissue extracts containing fractions relatively enriched for both PrPSc and PrP, after which the need to remove the PrP protein with a proteinase K treatment. This procedure entails at least 10 separate incubation and separation steps in which the absolute amount of the proteins to be detected in the sample is reduced at every step. Although this protocol works very well for the diagnosis of the clinical phase of SE's, where an abundance of PrPSc is present in relation to the normal cellular isoform PrP, in the preclinical phase of TSE, the absolute amount of PrPSc is so small that it usually gets lost during the preparation.
In BSE, the situation differs from that of scrapie: on the one hand, results from mice-transmission experiments using different tissues of BSE affected cattle, may indicate that distribution of the BSE agent tissues outside the CNS is not as extensive as in the case of scrapie in sheep, on the other hand it may be that the mice used in the bio-assays are far less sensitive for BSE than for scrapie. Experimental transmission of BSE to mice only succeeded when brain material was used (Fraser et al., 1988; Fraser et al., 1990); mice inoculated with other materials, including spleen, semen, buffy coat, muscle, bone marrow and placenta remained healthy.
However, all above techniques other than bio-assays have in common that diagnosis of SEs can only be established in the clinical phase of the disease, often at autopsy only. Considering the fact that bio-assays are very slow, due to the very slow progress of the disease in the experimental animal that is used for the bio-assay as such, no methods are currently available that offer immediate diagnoses of SEs in a pre-clinical phase of the disease. Thus, although the average expert in diagnostic test development has currently a wealth of diagnostic techniques available to detect all kinds of proteins in biological samples, using monoclonal or polyclonal antisera in enzyme- or label-linked immunoassays, using techniques with or without enriching methods for the protein under study, no gold-standard is available to give guidance to the development of those diagnostic techniques that would be applicable in the case of pre-clinical diagnosis of prion disease. In other words, methods to establish sophisticated diagnostic tests are currently well known to the general expert in the field; the expert lacks, however, methods to establish the sensitivity and specificity of those sophisticated diagnostic tests due to the lack of a “gold standard”.